1) To better understand the conformational conversion of normal PrP to infectious pathological forms in prion diseases, we have studied the effects of mutations of the PrP molecule in the region of the central lysine cluster (residues 101-110). Our prior molecular modeling of PrPSc structure suggested that the lysines in this cluster would likely prevent PrPSc-like folding and amyloid formation in the absence of anionic cofactors. Furthermore, mutations of prolines 102 and 105 to leucines in humans is known to cause Gerstmann-Straussler-Scheincker syndrome, which results from inadequately controlled spontaneous conversion to PrP amyloids. By systematically mutating these proline and lysine residues and analyzing effects on the in vitro formation of PrP amyloid fibrils, we have demonstrated that these residues do indeed impede the formation of amyloid fibrils with extended protease-resistant cores like those of PrPSc. Thus, we propose that these residues within PrP may, in addition to playing potential roles in the as yet mysterious normal physiological function of PrP, be evolutionarily selected to reduce the risk of premature and uncontrollable conversion to pathological forms. 2) In collaboration with Dr. Richard Bessen at Colorado State University, we investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH) Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but following transmission to SGH, the incubation period of CKY CWD was &#8764;150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrPSc polypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrPSc differed for the CWD strains in a host-dependent manner. WST CWD produced PrPSc amyloid plaques in the brain of SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrPSc from WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrPSc properties between transgenic mice and SGH. These findings indicated that host-specific pathways that are independent of Prnp can alter the PrPSc conformation of certain prion strains, leading to changes in the biophysical properties of PrPSc, neuropathology, and clinical prion disease. Our role in this project was to measure the relative levels of prion-seeding activity in the brains of infected animals by RT-QuIC.